TWI473622B - Methods of administering anti-tnfα antibodies - Google Patents

Description

Method of administering an anti-TNFα antibody

Tumor necrosis factor alpha (TNFα) is a cytokine produced by several cell types, including single cells and macrophages, which was originally identified for its ability to induce tumors in certain mice (see, eg, Old, L. (1985) Science 230 :630-632). Subsequently, a factor called malignant protein associated with dysentery shows the same molecule as TNFα. TNFα is associated with the regulation of shock (see, for example, Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57 : 505-518; Beulter, B. and Cerami, A. (1989) Annu. Rev. Immunol 7 : 625-655). Furthermore, TNFα has been linked to various human diseases and disorders, including sepsis, infection, autoimmune diseases, transplant rejection, and limb-to-host disease (see Vasilli, P. (1992) Annu. Rev. Immunol. 10 :411 -452; Tracey, KJ and Cerami, A. (1994) Annu. Rev. Med. 45 : 491-503).

Due to the deleterious role of human TNF[alpha] (hTNF[alpha]) in various human disorders, therapeutic targets have been designed to inhibit or counteract hTNF[alpha] activity. In particular, antibodies that bind to and neutralize hTNFα have been recognized as a means of inhibiting hTNFα activity. Some of the earliest antibodies were mouse monoclonal antibodies (mAbs) secreted by fusion tumors prepared from lymphocytes of mice immunized with hTNFα (see, for example, Hahn T., et al. (1985) Proc Natl Acad Sci USA 82 :3814 -3818; Liang, CM., et al. (1986) Biochem. Biophys. Res. Commun. 137 : 847-854; Hirai, M., et al. (1987) J. Immunol. Methods 96 : 57-62; Fendly, BM, et al. (1987) Hybridoma 6 :359-370; Moller, A., et al. (1990) Cytokine 2 : 162-169; Moeller et al., USP 5, 231, 024; Wallach, D. European Patent Application 186 833 B1 European Patent Application 218 868 A1 to Old et al.; European Patent Application 260 610 B1) to Moeller, A. et al. Although these mouse anti-hTNFα antibodies often exhibit high affinity for hTNFα (eg, K _d ≦10 ^-9 M) and can neutralize hTNFα activity, their in vivo use is limited by problems associated with human administration of mouse antibodies. For example, short serum half-life, the method of restricting certain human effector functions and inducing unwanted immune responses to mouse antibodies in humans ("human anti-mouse antibody" (HAMA) response).

In an attempt to overcome the use of whole mouse antibodies in humans, mouse anti-hTNFα antibodies are genetically engineered to be more "human-like". For example, chimeric antibodies in which a variable region of an antibody chain derived from a mouse and an antibody chain is derived from a human has been prepared (Knight, DM, et al. (1993) Mol. Immunol. 30 : 1443-1453; Daddona, PE, et al. PCT Application WO 92/16553). In addition, highly variable regions in which the variable regions of antibodies have been prepared have been derived from mice but other variable regions and antibody constant regions are derived from humanized human antibodies (Adair, JR, et al. PCT Application No. WO 92/ 11383). However, since these chimeric and humanized antibodies still leave a few mouse sequences, an unwanted immune response (human anti-chimeric antibody (HACA) response) can be induced, especially for chronic indications such as rheumatoid arthritis. For long-term administration (see, for example, Elliott, MJ, et al. (1994) Lancet 344 : 1125-1127; Elloit, MJ, et al. (1994) Lancet 344 : 1105-1110).

A preferred hTNF[alpha] inhibitor (e.g., a chimeric or humanized antibody) to a mouse mAbs or a derivative thereof is required to be an entire human anti-hTNF[alpha] antibody since this agent should not elicit the HAMA response even for prolonged use. Human monoclonal antibodies against hTNF[alpha] have been prepared using human fusion tumor technology (Boyle, P., et al. (1993) Cell. Immunol. 152 : 556-568; Boyle, P., et al. (1993) Cell. Immunol. 152 : 569-581; European Patent Publication 614 984 A2) by Boyle et al. However, these monoclonal antibodies derived from fusion tumors have been reported to have too low affinity for hTNFα to be calculated by conventional methods, unable to bind soluble hTNFα and are unable to neutralize hTNFα-induced cytotoxicity (see Boyle et al., supra). literature). Furthermore, the success of human fusion tumor technology relies on the presence of lymphocytes in the peripheral blood of humans that produce autoantibodies specific for hTNFα. Some studies have detected anti-hTNFα serum autoantibodies in human individuals (Fomsgaard, A., et al. (1989) Scand. J. Immunol. 30 : 219-223; Bendtzen, K., et al. (1990) Prog. Leukocyte Biol. 10B : 447-452), while others are absent (Leusch, HG., et al. (1991) J. Immunol. Methods 139: 145-147).

Another natural human anti-hTNFα antibody is a recombinant hTNFα antibody. Have been described at a relatively low affinity (e.g., K _d about 10 ^-7 M) and hTNFα binding with fast dissociation rate (K _off i.e. about ^{10-2 sec-1)} of the recombinant human antibodies (Griffiths, AD, et al. ( 1993) EMBO J. 12 : 725-734). However, due to their relatively rapid dissociation kinetics, such antibodies are not suitable for therapeutic use. Furthermore, recombinant human anti-hTNFα has been described to be unable to neutralize hTNFα activity, but it enhances hTNFα binding to the cell surface and enhances the integration of hTNFα (Lidburg, A., et al. (1994) Biotechnol. Ther. 5 : 27-45; Aston, R., et al., PCT Application No. WO 92/03145).

Recombinant human antibodies that bind to soluble hTNFα with high affinity and reduced kinetics and have neutralizing hTNFα activity (including hTNFα-induced cytotoxin and hTNFα-induced cell activation in vitro and in vivo) are also described (see USP 6,090,382). The typical mode of administration of antibodies is performed intravenously weekly. Administration of antibodies and/or any drug per week may be costly, inconvenient, and add several side effects due to the number of administrations. Intravenous administration also has a limitation that administration by a drug trainer is generally performed.

The present invention provides a biweekly route of treatment for dysregulation associated with TNFα, preferably by subcutaneous route. Biweekly administration has many advantages over weekly administration, including (but not limited to) fewer total injections, reduced number of injection site reactions (such as localized pain and swelling), increased patient compliance (due to less The number of injections) and the cost of the patient and the care provider. The advantage of subcutaneous administration is that the patient itself can be administered a therapeutic substance, such as a human TNFα antibody, which is convenient for both the patient and the care provider.

The present invention provides a method of treating disorders in which TNFα activity is detrimental. The method comprises administering to the individual a biweekly subcutaneous injection of the antibody. Preferably, the antibody is a recombinant human antibody that specifically binds to human TNFα. The invention further provides a method of treating a disorder in which TNFα activity is detrimental. Such methods include the use of combination therapies in which a human antibody is administered to an individual with other therapeutic agents, such as one or more other antibodies that bind to other targets (eg, antibodies that bind to other cytokines or bind to cell surface molecules), one or more cells. , soluble TNFα receptor (see, for example, PCT Patent Publication WO 94/06476) and/or one or more chemical agents that inhibit hTNFα production or activity (such as cyclohexyl-based derivatives as described in PCT Publication WO 93/19751) ()), better methotrexate. Preferably, the antibody is a recombinant human antibody that specifically binds to human TNFα. The antibody of the present invention is characterized by binding to hTNFα with high affinity and reduced kinetics and neutralizing hTNFα activity, including hTNFα-induced cytotoxicity (in vitro and in vivo) and hTNFα-induced cell activation. The antibody may be full length (such as an IgGl or IgG4 antibody) or may include only antigen-binding sites (such as Fab, F(ab') ₂ , scFv fragments or a single region). The optimal recombinant antibody of the invention is referred to as D2E7, having a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3 and a heavy amino acid sequence comprising SEQ ID NO: 4 (see Appendix B). chain. Preferably, the D2E7 antibody has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain (HCVR) comprising the amino acid sequence of SEQ ID NO: 2. Such antibodies are described in USP 6,090,382, which is incorporated herein by reference.

In one embodiment, the invention provides a method of treating a disorder in which TNFα activity is detrimental. Such methods include inhibiting human TNF[alpha] activity by subcutaneous administration of an anti-TNF[alpha] antibody in two weeks and thus treating the disorder. The disorder may be, for example, septicemia, autoimmune diseases (such as rheumatoid arthritis, allergies, multiple sclerosis, autoimmune diabetes, autoimmune uveitis, and nephrotic syndrome), infectious diseases, cachexia, transplant rejection, or grafting. Limb-to-host disease, lung disorders, bone disorders, intestinal disorders, or heart disorders.

In another embodiment, the invention provides a method of treating a disorder in which TNFα activity is detrimental. The methods include subcutaneous administration of an anti-antibiotic to an individual for two weeks. The TNFα antibody and methotrexate inhibit human TNFα activity and thus treat the disorder. In one aspect, methotrexate is administered prior to administration of the anti-TNFα antibody. In another aspect, methotrexate is administered after administration of the anti-TNFα antibody.

In a preferred embodiment, the anti-TNFα antibody for treating a disorder in which TNFα activity is detrimental is a human anti-TNFα antibody. Even more preferably, the treatment can be administered subcutaneously to the isolated human antibody or antigen-binding site thereof in two weeks. Preferably, the antibody or antigen-binding site thereof is cleaved from human TNFα with a K _d of 1×10 ^-8 M or less and a _Koff rate constant of 1×10 ⁻³ s ⁻¹ or less (both by surface cell plasmid) genomic resonance assay) and analytical standards for in vitro L929 to the 1 × 10 ^-7 M or less of IC ₅₀ and cytotoxicity of human TNFα. Better isolated human antibody or antigen-binding sites in the K _off of less or 5 × 10 ^-4 s ^-1 or even more to 1 × 10 ^-4 s ^-1 or less of the K _off isolated from human TNFα. Better, the human antibody or antigen isolated - A ₅₀ L929 vitro binding site and analysis of the following criteria or to IC 1 × 10 ^-8 M _50, and even better 1 × 10 ^-10 M or less of IC Human TNFα cytotoxicity.

In another embodiment, the present invention provides a method of treating a disorder in which TNFα activity is detrimental, by administering a human antibody or an antigen binding site thereof subcutaneously to an individual for two weeks. The antibody or antigen-binding site thereof preferably has the following characteristics: a) dissociation from human TNFα by K _off of surface cell plasmid genomic resonance of 1 × 10 ^-3 s ^-1 or less; b) having an amine including SEQ ID NO: The acid sequence is substituted at position 1, 4, 5, 7 or 8 or by 1 to 5 conservative amino acids at position 1, 3, 4, 6, 7, 8 and/or 9 by a single alanine substitution. SEQ ID NO: 3 modified light chain CDR3 region; c) having an amino acid sequence comprising SEQ ID NO: 4 or substituted with a single alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or The heavy chain CDR3 region modified from SEQ ID NO: 4 at position 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12 is substituted with 1 to 5 conservative amino acids.

More preferably, the antibody or antigen-binding site thereof is cleaved from human TNFα with a _Koff of 5 × 10 ^-4 s ^-1 or less. Still more preferably, the antibody or antigen-binding site thereof is cleaved from human TNFα with a _Koff of 1 × 10 ^-4 s ^-1 or less.

In yet another embodiment, the invention provides a method of treating a disorder in which TNFα activity is detrimental. Such methods include subcutaneous administration of a human antibody or antigen binding site thereof to an individual for two weeks. Preferably, the antibody or antigen-binding site thereof comprises LCVR having a CDR3 region comprising SEQ ID NO: 3 or a CDR3 region modified by SEQ ID NO: 3 at position 1, 4, 5, 7 or 8 by a single alanine. An HCVR having a CDR3 region comprising SEQ ID NO: 4 or a CDR3 region modified from SEQ ID NO: 4 at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 by a single alanine. More preferably, the LCVR further has a CDR2 region comprising the amino acid sequence of SEQ ID NO: 5 and the CDR2 region of the HCVR comprising the amino acid sequence of SEQ ID NO: 6. Still more preferably, the LCVR further has a CDR1 region comprising the amino acid sequence of SEQ ID NO: 7 and the HCVR has a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8.

In still another embodiment, the present invention provides a method of treating a disorder in which TNFα activity is detrimental, by administering an individual human antibody or antigen-binding site thereof subcutaneously to a subject for two weeks. The antibody or antigen-binding site thereof preferably comprises an HCVR having an amino acid sequence comprising SEQ ID NO: 1 and an HCVR comprising the amino acid sequence of SEQ ID NO: 2. In a specific example, the antibody has an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In yet another embodiment, the antibody is a Fab fragment, an F(ab') ₂ fragment, or a single chain Fv fragment.

In still another embodiment, the invention provides a method of treating a disorder in which an anti-TNFα antibody is administered to an individual, wherein one or more anti-TNFα antibodies or antigen-binding sites thereof are administered subcutaneously to the individual. Preferably, the antibody or antigen-binding portion thereof comprises SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18. The LCVR of the CDR3 region of the amino acid sequence of the ensemble number 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 CDR3 of the amino acid sequence of the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35 HCVR of the area.

Another object of the invention is a kit comprising a formulation comprising a pharmaceutical composition. The kit includes an anti-TNFα antibody and a pharmaceutically acceptable carrier. The kit contains a copy indicating that the pharmaceutical composition is administered subcutaneously for two weeks to treat a disorder in which the anti-TNFa antibody is administered. In another aspect, the invention relates to a kit comprising a formulation comprising a pharmaceutical composition and further comprising an anti-TNFα antibody, methotrexate, and a pharmaceutically acceptable carrier. The kit contains a copy indicating that the pharmaceutical composition is administered subcutaneously for two weeks to treat a disorder in which the anti-TNFa antibody is administered.

A further object of the invention is to provide a preloaded syringe comprising a pharmaceutical composition comprising an anti-TNFa antibody and a pharmaceutically acceptable carrier. In yet another object, the invention provides a preloaded syringe comprising a pharmaceutical composition comprising an anti-TNFα antibody, methotrexate, and a pharmaceutically acceptable carrier.

The present invention relates to a method of treating a disorder in which administration of anti-TNFα is beneficial, comprising administering a single human antibody or antigen-binding site thereof which binds to human TNFα with high affinity, low dissociation rate and high neutralizing ability, thereby treating the Disorder. Various objects of the invention relate to the treatment of such antibodies and antibody fragments and pharmaceutical compositions thereof.

In order to better understand the present invention, certain nouns are first defined.

As used herein, "administering" means administering a substance (such as an anti-TNFα antibody) to achieve a therapeutic target (eg, treating a TNFα-related disorder).

As used herein, "biweekly medication", "biweekly" and "biweekly administration" represent the time-lapse of a subject (eg, anti-TNFα antibody) to achieve a therapeutic target (eg, treatment of TNFα-related disorders). process. Biweekly dosing regimens do not intend to include weekly dosing regimens. Preferably, the substance is administered every 9-19 days, preferably every 11-17 days, even better every 13-15 days and preferably every 14 days.

As used herein, "combination therapy" refers to the administration of two or more therapeutic substances such as anti-TNFα antibodies and the drug methotrexate. The methotrexate can be administered simultaneously with the anti-TNFα antibody, before or after the anti-TNFα antibody.

As used herein, "human TNFα" (abbreviated herein as hTNFα or reduced to hTNF) is intended to represent a 17 kD secretory state and a 26 kD membrane-bound state (a biologically active state consisting of a non-covalently bound 17 kD molecule terpolymer) Human cytokines present. The structure of TNFα is described, for example, in Pennica, D., et al. (1984) Nature 312 : 724-729; Davis, JM, et al. (1987) Biochemistry 26 : 1322-1326; and Jones, EY, et al. (1989) Nature. 338 : 225-228. The term human TNF[alpha] is intended to include recombinant human TNF[alpha], which can be prepared by standard recombinant expression methods or commercially available (R&D Systems, Catalog No. 210-TA, Minneapolis, MN).

As used herein, "antibody" is intended to mean an immunoglobulin molecule composed of four polypeptide chains (two heavy (H) chains and two light (L) chains cross-linked by disulfide bonds). Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three regions: CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one region CL. The VH and VL regions are in turn subdivided into highly variable regions (referred to as complementarity determining regions (CDRs)) that are inter-configured with regions of more conservative network regions (FR). Each of the VH and VL lines is composed of three CDRs and four FRs, and is arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

An "antigen binding site" (or simply "antibody site") of an antibody as used herein refers to one or more fragments (eg, hTNFα) of an antibody that retains the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be carried out by a fragment of a full length antibody. Examples of the binding fragment contained in the "antigen-binding site" of the antibody include (i) a Fab fragment, a monovalent fragment composed of VL, VH, CL, and CH1 regions; (ii) a F(ab') ₂ fragment, which is composed of a disulfide. a bridge linking a bivalent fragment of two Fab fragments in the hinge region; (iii) an Fd fragment consisting of a VH and a CH1 region; (iv) an Fv fragment consisting of a VL and VH region of the one-arm of the antibody; (v) a dAb fragment (Ward et al. (1989) Nature 341 :544-546) consisting of a VH region; and (vi) an individual complementarity determining region (CDR). Furthermore, although the two regions VL and VH of the Fv fragment are encoded by individual genes, they can be recombined to form a synthetic bond in which a single protein chain of VL and VH regions are paired to form a monovalent molecule. (referred to as single chain Fv (scFv); see, eg, Bird et al. (1988) Science 242 : 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 5879-5883). This single chain antibody is also intended to be included in the term "antigen binding site" of the antibody. Other types of single chain antibodies such as diabodies are also included. A diabodies are bivalent dispecific antibodies that display VH and VL regions on a single polypeptide chain, but the use of too short linkages does not allow pairing between the two regions of the same chain, thereby driving the region to complementary regions with other chains. Pairing and generating two antigen binding positions (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90 :6444-6448; Poljak, RJ, et al. (1994) Structure 2 : 1121- 1123).

Further, the antibody or antigen binding site thereof can be part of a larger immunoadhesive molecule formed by covalent or non-covalent association of the antibody or antibody site with one or more other proteins or peptides. An example of such an immunoadhesive molecule comprises the use of a streptavidin core region to produce a tetrameric scFv molecule (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6 : 93-101) and the use of cysteine residues, marker peptides and The C-terminal polyglycolic acid tag produces bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31 : 1047-1058). Antibody sites such as Fab and F(ab') ₂ fragments can be prepared from whole antibodies using conventional techniques, such as whole antibodies being digested with papain or pepsin, respectively. Furthermore, antibodies, antibody sites and immunoadhesive molecules can be obtained using standard recombinant DNA techniques as described herein.

As used herein, "human antibody" is intended to include antibodies having variable and constant regions and derived from human germline immunoglobulin sequences. The human antibody of the present invention may comprise an amino acid residue but is not encoded by a human germline immunoglobulin sequence (such as a random or position-specific genetic mutation in vitro or a mutation introduced by somatic mutation in vivo), for example In the CDRs and in the specific CDR3. However, "human antibody" as used herein does not intend to include antibodies in which CDR sequences derived from other mammalian species, such as mouse germlines, have been grafted to human framework sequences.

As used herein, "recombinant human antibody" is intended to include all human antibodies that are prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (described in paragraph II below). An antibody isolated from a recombinant human antibody gene pool (described in detail in paragraph III below), an isolated antibody from an animal (such as a mouse) that has a gene transfer effect on a human immunoglobulin gene (see, for example, Taylor, LD). , et al. (1992) Nucl. Acids Res. 20 : 6287-6295) or an antibody prepared, expressed, produced or isolated by any other means involving the cleavage of human immunoglobulin gene sequences into other DNA sequences. This recombinant human antibody has variable and constant regions derived from human germline immunoglobulin sequences. However, in a specific example, the recombinant human antibody undergoes in vitro gene mutation (or when the somatic gene mutation in vitro uses an animal that transfers gene to the human Ig sequence) and thus the amino acid sequence of the VH and VL regions of the recombinant antibody, Although derived from and associated with human germline VH and VL sequences, sequences that are not naturally found in the human antibody germline library in vivo.

As used herein, "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies having different antigenic specificities (eg, a single antibody that specifically binds hTNF[alpha] is substantially free of antibodies that specifically bind to an antigen other than hTNF[alpha]). However, an isolated antibody that specifically binds hTNFα may have cross-reactivity to other antigens, such as hTNFα molecules from other species (discussed later). Furthermore, the isolated antibodies are substantially free of other cellular materials and/or chemicals.

As used herein, "neutralizing antibody" (or "antibody capable of neutralizing hTNFα activity") is intended to represent an antibody which binds to hTNFα and which causes inhibition of hTNFα biological activity. The inhibition of the biological activity of hTNFα can be assessed by measuring one or more indicators of the biological activity of hTNFα, such as hTNFα-induced cytotoxicity (in vitro or in vivo), hTNFα-induced cell activation, and hTNFα binding to the hTNFα receptor. Such indicators of hTNF[alpha] biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see Example 4). Preferably, the ability of the antibody to neutralize hTNFα activity is assayed by inhibition of hTNFα-induced cytotoxicity of L929 cells. As for other or additional parameters of hTNFα, the ability of the antibody to inhibit hTNFα-induced expression of ELAM-1 on HUVEC (an indicator of hTNFα-induced cell activation) can be analyzed.

As used herein, "surface plasmid cell genomic resonance" means that the actual time biospecific interaction can be analyzed by, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Switzerland and Pickup, NJ) to detect changes in protein concentration in the biosensor matrix. Optical performance. For further description see Example 1 and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51 : 19-26; Jonsson, U., et al. (1991) Biotechniques 11 : 620-627; Johnsson, B., Et al. (1995) J. Mol. Recognit. 8 : 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198 :268-277.

The term " _Koff " as used herein is intended to mean the rate of detachment of the antibody from the antibody/antigen complex.

As used herein the "K _d" word to be representative of a specific antibody - antigen interaction of the dissociation constant.

As used herein, a "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably a double-stranded DNA.

As used herein, a "isomeric nucleic acid molecule" for use in a nucleic acid encoding an antibody or antibody protein (eg, VH, VL, CDR3) that binds to hTNFα is intended to represent a nucleotide sequence encoding the antibody or antibody site that does not contain a binding to hTNFα. A nucleic acid molecule of an antibody other than the antigen or other nucleotide sequence encoded by the antibody site, the other sequence being flanked by nucleic acids in the human genomic DNA. Thus, for example, an isolated nucleic acid encoding a VH region of an anti-hTNFα antibody of the invention does not contain additional sequences encoding other VH regions that bind to an antigen other than hTNFα.

As used herein, "vector" is intended to mean a nucleic acid molecule that can carry other nucleic acids to which it is linked. One type of vector is a "plastid" which represents a circular double stranded DNA loop in which additional DNA fragments can be ligated. Another type of vector is a viral vector. Additional DNA fragments can be ligated into the viral genome. Certain vectors are capable of spontaneous replication in a host cell into which they are introduced (e.g., a bacterial vector having a bacterial region of replication and a free gene mammalian vector). Other vectors (such as non-free gene mammalian vectors) can be integrated into the genome of the host cell by introduction into the host cell and thus replicated along with the host genome. Furthermore, certain vectors can direct the expression of a gene to its operably linked. This vector is referred to herein as a "recombinant expression vector" (or simply "expression vector"). In general, the utility of expression vectors in recombinant DNA techniques is often in a qualitative state. In this specification, "plastid" and "carrier" are used interchangeably because plastid is the most commonly used type of carrier. However, the present invention is intended to encompass other types of expression vectors such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and gland-associated viruses) that have equivalent functions.

As used herein, "recombinant host cell" (or simply "host cell") is intended to mean a cell into which a recombinant expression vector has been introduced. It is to be understood that this term is not only intended to represent a particular individual cell but also to represent the descendants of this cell. Since some modifications can occur in the subculture due to mutation or environmental influences, this offspring is in fact different from the mother cell, but is still included in the scope of the "host cell" used herein.

Various objects of the invention are further illustrated in the following paragraphs.

I. Human antibodies that bind to human TNFα

The present invention provides a method of treating a disorder in which administration of anti-TNFa is beneficial. The method comprises biweekly subcutaneous administration of a single human antibody or antigen binding site thereof which binds to human TNFα with high affinity, low dissociation rate and high neutralizing ability. Preferably, the human antibody of the present invention is recombinant neutralizing human anti- hTNFα antibody. The optimal recombinant neutralizing antibody of the invention is referred to herein as D2E7 (the amino acid sequence of the D2E7 VL region is shown in SEQ ID NO: 1; the amino acid sequence of the D2E7 VH region is shown in SEQ ID NO: 2). The nature of D2E7 is described in Salfeld et al., U.S. Patent No. 6,090,382, the disclosure of which is incorporated herein by reference.

In one aspect, the invention provides a method of treating a disorder in which administration of anti-TNFa is beneficial. The method comprises subcutaneously administering D2E7 antibody and antibody site, D2E7-related antibody and antibody site and other human antibodies and antibody sites equivalent to D2E7, such as high affinity binding to hTNFα, low dissociation kinetics and high-neutrality. Those who can. In a specific embodiment, the present invention provides treatment with a single human antibody or an antigen binding site thereof, which can be 1 × 10 ^-8 M or less K _d and 1 × 10 ^-3 s. K _{off of} ^-1 or below was dissociated from human TNFα (both determined by surface plasmid cell genomic resonance) and human TNFα cytotoxicity was neutralized with an IC ₅₀ of 1×10 ^-7 M or less in the in vitro L929 assay standard. The better isolated human antibody or antigen-binding sites of the following or may be 5 × K _off 10 ^-4 s ^-1, or even better than 1 × 10 ^-4 s ^-1 or less of the K _off TNFα from human Solutions off. The better isolated human antibody or antigen-binding sites in vitro L929 or analysis of the following criteria to IC 1 × 10 ^-8 M _50, and even better 1 × 10 ^-9 M or less and more of IC ₅₀ and 1 The IC ₅₀ of ×10 ^-10 M or less neutralizes human TNFα cytotoxicity. In a preferred embodiment, the antibody is an isolated human recombinant antibody or an antigen binding site thereof.

It is well known to those skilled in the art that antibody heavy and light chain CDR3 regions play an important role in the binding specificity/affinity of antibodies to antigens. Accordingly, in another aspect, the present invention relates to a method of treating a disorder in which an anti-TNFα antibody is administered, which is administered subcutaneously with a light-reducing kinetics in combination with TNFα and having a structurally identical or related D2E7 structure. Human antibodies in the CDR3 region of the plastid and heavy. Position 9 of the D2E7 VL CDR3 can be occupied by Ala or Thr without substantially affecting the K _off . Accordingly, the conserved motif of the D2E7 VL CDR3 includes the amino acid sequence: QRYNRAPY-(T/A) (SEQ ID NO: 3). In addition, the position 12 of the D2E7 VH CDR3 can be occupied by Tyr or Asn without substantially affecting the K _off . Accordingly, the conserved motif of the D2E7 VH CDR3 includes the amino acid sequence: VSYLSTASSLD-(Y/N) (SEQ ID NO: 4). Furthermore, as demonstrated in Example 2, the CDR3 region of the D2E7 heavy and light chain was modified to be replaced with a single alanine residue (position 1, 4, 5, 7 or 8 within the VL CDR3 or position 2 within the VH CDR3) , 3, 4, 5, 6, 8, 9, 10 or 11) without substantially affecting the K _off . Still better known to those skilled in the art, it will be apparent that assuming that the D2E7 VL and VH CDR3 regions are modified by alanine substitution, substitution of other amino acids in the CDR3 region is possible and still maintains a low dissociation rate constant of the antibody, especially with a conservative amino acid. Replace. As used herein, "conservative amino acid substitution" is one in which one of the amino acid residues is substituted with another amino acid residue having a similar side chain. A family of amino acid residues having similar side chains are defined in the art and include basic side chains (eg, amino acid, arginine, histidine), acidic side chains (eg aspartic acid, hydrazine). Amino acid), uncharged polar side chain (eg glycine, aspartame, amidoxime, serine, threonine, tyrosine, cysteine), non-polar side chain (eg propylamine) Acid, valine, leucine, isoleucine, valine, phenylalanine, methionine, tryptophan), β-branched side chains (such as threonine, valine, leucovorin) Aminic acid) and aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Preferably no more than one to five conservative amino acid substitutions are made in the D2E7 VL and/or VH CDR3 regions. More preferably, no more than one to three conservative amino acid substitutions are made in the D2E7 VL and/or VH CDR3 regions. In addition, conservative amino acid substitutions are not made at positions of amino acids that are important for binding to hTNFα. Positions 2 and 5 of D2E7 VL CDR3 and positions 1 and 7 of D2E7 VH CDR3 appear to be important for interaction with hTNFα, therefore, conservative amino acid substitutions are better not performed at this position (but as will be described later, D2E7 VL CDR3 position 5 The alanine substitution is acceptable) (see USP 6,090,382).

Accordingly, in another embodiment, the present invention relates to a method of treating a disorder in which an anti-TNFα antibody is administered, which is administered subcutaneously to an isolated human antibody or antigen-binding site thereof. The antibody or antigen-binding site thereof preferably has the following characteristics: a) dissociation from human TNFα by K _off of surface cell plasmid genomic resonance of 1 × 10 ^-3 s ^-1 or less; b) having an amine including SEQ ID NO: The acid sequence is substituted at position 1, 4, 5, 7 or 8 or by 1 to 5 conservative amino acids at position 1, 3, 4, 6, 7, 8 and/or 9 by a single alanine substitution. SEQ ID NO: 3 modified light chain CDR3 region; c) having an amino acid sequence comprising SEQ ID NO: 4 or substituted with a single alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or The heavy chain CDR3 region modified from SEQ ID NO: 4 at position 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12 is substituted with 1 to 5 conservative amino acids.

More preferably, the antibody or antigen-binding site thereof is cleaved from human TNFα with a _Koff of 5 × 10 ^-4 s ^-1 or less. Still more preferably, the antibody or antigen-binding site thereof is cleaved from human TNFα with a _Koff of 1 × 10 ^-4 s ^-1 or less.

In yet another embodiment, the invention provides a method of treating a disorder in which an anti-TNFa antibody is administered. Such methods include subcutaneous administration of isolated human antibodies or antigen binding sites thereof in a biweekly manner. Preferably, the antibody or antigen-binding portion thereof comprises a light-weight CDR3 region having the amino acid sequence comprising SEQ ID NO: 3 or substituted with a single alanine at position 1, 4, 5, 7 or 8 and modified from SEQ ID NO: Chain variable region (LCVR) and having an amino acid sequence comprising SEQ ID NO: 4 or modified by single alanine at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 from sequence number 4 Heavy chain variable region (HCVR) of the CDR3 region. More preferably, the LCVR further has a CDR2 region comprising the amino acid sequence of SEQ ID NO: 5 (i.e., D2E7 VL CDR2) and the HCVR further has a CDR2 region comprising the amino acid sequence of SEQ ID NO: 6 (i.e., D2E7 VH CDR2). Even more preferably, the LCVR further has a CDR1 region comprising the amino acid sequence of SEQ ID NO: 7 (i.e., D2E7 VL CDR1) and HCVR having a CDR1 region comprising the amino acid sequence of SEQ ID NO: 8 (i.e., D2E7 VH CDR1). VL framework regions of V _K I is preferably derived from human germline family, more preferably a gene derived from human germline Vk A20 and the line 1A and 1B of the D2E7 VL framework sequences shown in USP 6,090,382 are preferably derived from the FIG. Preferably the VH framework region from a human V _H 3 germline family, more preferably from DP-31 human germline VH gene and 2A and 2B of the D2E7 VL framework sequences shown in USP 6,090,382 are preferably derived from the FIG.

In still another embodiment, the present invention provides a method of treating a disorder in which an anti-TNFα antibody is administered, which is administered subcutaneously to a human antibody or antigen-binding site thereof. Preferably, the antibody or antigen binding site thereof comprises a light chain having an amino acid sequence comprising SEQ ID NO: 1. Variable region (LCVR) (i.e., D2E7 VL) and heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 (i.e., D2E7 VH). In a specific example, the antibody comprises a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Furthermore, the antibody may comprise a light chain constant region, which is a kappa light chain constant region or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody can be a Fab fragment or a single chain Fv fragment.

In still another embodiment, the present invention provides a method of treating a disorder in which an anti-TNFα antibody is administered to an individual, which is administered subcutaneously to an isolated human antibody or antigen-binding site thereof. Preferably, the antibody or antigen binding site thereof comprises a D2E7-related VL and VH CDR3 region, such as an antibody or antigen binding site thereof, which comprises a sequence selected from SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14. Sequence number 15, sequence number 16, sequence number 17, sequence number 18, sequence number 19, sequence number 20, sequence number 21, sequence number 22, sequence number 23, sequence number 24, sequence number 25, and sequence number 26 The LCVR of the CDR3 region of the amino acid sequence of the group includes or is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, The HCVR of the CDR3 region of the amino acid sequence of the group of SEQ ID NO: 34 and SEQ ID NO: 35.

An antibody or antibody site thereof of the invention can be derivatized or linked to another functional molecule (such as another peptide or protein). Accordingly, the antibody or antibody of the present invention The site is intended to comprise the derivatized and other modified human anti-hTNFα antibodies herein, including immunoadhesive molecules. For example, an antibody or antibody site of the invention may be functionally linked (by chemical coupling, gene fusion, non-covalent association or otherwise) to one or more other molecular entities, such as another antibody (eg, a bispecific antibody or a diabodies), A detectable agent, a cytotoxic agent, a medicinal agent, and/or a protein or peptide (eg, a streptavidin core region or a polyhistidine tag) that can modulate the antibody or antibody site with another molecule.

One type of derivatized anti-system is made by cross-linking two or more antibodies (of the same type or different types, such as the production of bispecific antibodies). Suitable crosslinkers comprise heterofunctional or homobifunctional groups having two different reactive groups separated by a suitable spacer such as m-maleimidobenzylidene-N-hydroxysuccinimide. (such as adiponectin azelate). This linkage was obtained from Pierce Chemical Company, Rockford, IL.

Useful detectable agents that can be used to derivatize an antibody or antibody site of the invention comprise a fluorescent compound. The fluorescent detectable agent is exemplified by fluorescein, fluorescein isothiocyanate, basic ruthenium red, 5-dimethylamine-1-naphthalene sulfonium chloride, phycobilicilin and the like. Antibodies can also be used to detect enzyme derivatization, such as alkaline phosphate, horseradish peroxidase, glucose oxidase and the like. When an antibody is derivatized with a detectable enzyme, the addition of an enzyme produces an additional reagent that detects the reaction product. For example, when a detectable agent, horseradish peroxidase, is present, the addition of cyanide and diaminobenzidine results in a colored reaction product that can be detected. Antibodies can also be biotinylated and detected by indirect measurement of avidin or streptavidin binding.

II. Antibody performance

The antibody or antibody site of the present invention can be prepared by recombinantly expressing the immunoglobulin light and heavy chain genes in a host cell. For recombinant expression of the antibody, the host cell is transfected with one or more recombinant expression vectors carrying a DNA fragment encoding the immunoglobulin light and heavy chain of the antibody, thereby allowing the light and heavy chains to be expressed in the host cell, preferably The antibody is secreted into a medium in which the host cells are cultured, and the antibody is recovered from the medium. Standard recombinant DNA methods are used to obtain antibody heavy and light chain genes, which are incorporated into recombinant expression vectors and introduced into host cells, such as Sambrook, Fritsch and Maniatis (editor), molecular selection; laboratory manuals , 2nd ed., New York, Cold Spring Harbor Press (1989), Ausubel, FM, et al. (ed.) Existing Strategies in Molecular Biology, Greene Publishing Association (1989) and USP 4,816,397 to Boss et al.

To express a D2E7 or D2E7-related antibody, a DNA fragment encoding a variable region of a light and heavy chain is first obtained. These DNAs can be obtained by polymerase chain reaction (PCR) amplification and modification of germline light and heavy chain variable sequences. The germline DNA sequences of human heavy and light chain variable region genes are known in the art (see, for example, the "Vbase" human germline sequence database; see also Kabat, EA et al. (1991) Immunologically interesting protein sequences, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al. (1992) "Human germline _VH sequence collection reveals _VH segments 5th group with different highly variable loops J. Mol. Biol. 227 :776-798 and Cox, JPL, et al. (1994) "Management of human germline V ₇₈ fragments revealed a strong basis for its treatment" Eur. J. Immunol. 24 : 827-836 ; its content is for reference in this article). To obtain a DNA fragment encoding a heavy chain variable region of a D2E7 or D2E7-related antibody, VH3 family members of the human germline VH gene were amplified by standard PCR. Preferably, the DP-31 VH germline sequence is amplified. To obtain or light chain encoding D2E7-related antibody D2E7- DNA fragment of the variable region, V _K VL human germline gene family member I by standard PCR amplification. Preferably, the A20 VL germline sequence is amplified. PCR primers suitable for amplifying the DP-31 germline VH and A20 germline VL sequences can be designed using standard methods based on the nucleotide sequences disclosed in the references cited in the above references.

When a germline VH and VL fragment is obtained, the sequences can be mutated to encode a D2E7 or D2E7-related amino acid sequence disclosed herein. The amino acid sequence encoded by the germline VH and VL DNA sequences is first compared to the D2E7 or D2E7-related VH and VL amino acid sequences to identify amino acid residues other than the germline D2E7 or D2E7-related sequences. Next, the appropriate nucleotide mutation of the germline DNA sequence allows the mutated germline sequence to encode a D2E7 or D2E7-related amino acid sequence using a genetic code to determine the nucleotide change to be made. The gene mutation of the germline sequence is carried out by standard methods, such as PCR-regulated gene mutation (in which the mutated nucleotide is incorporated into the PCR primer such that the PCR product contains the mutation) or gene mutation at the leader position.

When a DNA fragment encoding a D2E7 or D2E7-related VH and VL fragment is obtained (by the above-described germline VH and VL gene amplification and gene mutation), the DNA fragments can be manipulated by standard recombinant DNA techniques, for example, The region gene is converted into a full-length antibody chain gene and transformed into a Fab fragment gene or a scFv gene. In such operations, the VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or Flexible bond. As used herein, "operably linked" means that the two amino acid fragments are combined such that the amino acid sequence encoded by the two DNA fragments remains in the framework.

The isolated DNA encoding the VH region can be converted into a full length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule (CH1, CH2 and CH3) encoding a constant region of the heavy chain. Human heavy chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al. (1991) Immunologically interesting protein sequences, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242) And DNA fragments comprising such regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is preferably an IgGl or IgG4 constant region. In the case of a Fav fragment heavy chain gene, the VH-encoding DNA is operably linked to another DNA molecule encoding only the constant region of the heavy chain CH1.

The isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule CL encoding a constant region of the light chain. Human light chain constant region gene sequences are known in the art (see, for example, Kabat, EA, et al. (1991) Immunologically interesting protein sequences, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242) And DNA fragments comprising such regions can be obtained by standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but is preferably a kappa constant region.

To generate the scFv gene, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible link (eg, encoding an amino acid sequence (Gly ₄ -Ser) ₃ ) such that VH and VL Sequences can be expressed as continuous single-stranded proteins, while VL and VH regions are linked by flexible linkages (see, for example, Bird et al. (1988) Science 242 : 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci .USA 85 : 5879-5883; McCafferty et al, Nature (1990) 348 :552-554).

To express an antibody or antibody site of the invention, the DNA fragment encoding the portion or full-length light and heavy chain obtained above is inserted into an expression vector such that the gene is operably linked to the transcriptional and translational control sequences. In the present specification, "operably linked" means that the antibody is ligated to the vector such that the transcriptional and translational control sequences within the vector have the desired function of regulating the transcription and translation of the antibody gene. The expression vector and the expression control sequence are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into another vector, or more typically, the two genes are inserted into the same expression vector. The antibody gene is joined by standard methods (e.g., binding of the antibody gene fragment to the complementary restriction site of the vector or, if no restriction is present, the blunt end junction). The expression vector may already carry the constant region sequence of the antibody prior to insertion of the D2E7 or D2E7-related light or heavy chain sequence. For example, one of the methods for converting a D2E7 or D2E7-related VH and VL sequence into a full-length antibody gene is to insert it into a expression vector that has separately encoded a heavy chain constant region and a light chain constant region, and thus the VH fragment is operably linked. The CH fragment and the VL fragment linked to the vector are operatively linked to the CL fragment in the vector. Additionally or alternatively, the recombinant expression vector can encode a single peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene is optionally incorporated into a vector such that a single peptide is linked in the framework to the amine terminus of the antibody chain gene. The single peptide can be an immunoglobulin single peptide or a single single Peptide (ie, a single peptide derived from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in the host cell. A "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (such as polyadenylation signals) that control the transcription or translation of the antibody chain genes. This regulatory sequence is described, for example, in Goeddel; Gene Expression Technology: Enzyme Methods 185, College Press, San Diego, CA (1990). It is well known in the art that the design of the expression vector includes the selection of regulatory sequences depending on several factors, the choice of host cell to be transformed, the degree of protein expression desired, and the like. Preferred regulatory sequences for mammalian host cells include viral elements that are introduced into mammalian cells to introduce high amounts of protein, such as promoters/enhancers derived from cytomegalovirus (CMV) (eg CMV promoter/enhancer), Simon (Simian) virus 40 (SV40) (eg SV40 promoter/enhancer), adenovirus (eg adenovirus major delayed promoter (AdMLP)) and polyomavirus. For further description of the viral regulatory elements and their sequences, see, for example, USP 5,168,062 to Stinski, USP 4,510,245 to Bell et al., and Schaffner et al. USP 4,968,615.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences (e.g., replication sources) and selectable marker genes that modulate vector replication in a host cell. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (see, for example, Axel et al., USP 4,399,216; 4,634,665 and 5,179,017). For example, a selectable marker gene confers resistance to a drug such as G418, hygromycin or methotrexate to a host cell into which the vector has been introduced. A preferred alternative marker gene comprises a dihydrofolate reductase (DHFR) gene (for dhfr ^- host cells with methotrexate selectivity/amplification) and a neo gene (for G418 selection).

For light and heavy chain performance, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. Various types of "transfection" are intended to encompass a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. Although it is theoretically possible to express the antibody of the present invention in a prokaryotic or eukaryotic host cell, it is preferred that eukaryotic cells, and particularly mammalian cells, are more likely to assemble and secrete appropriately folded and immunologically active antibodies than prokaryotic cells. Antibodies are expressed in cells, especially in mammalian cells. Prokaryotic expression of antibody genes has been reported to be ineffective in producing high yields of active antibodies (Boss, MA and Wood, CR (1985) Immunology Today 6 : 12-13).

Preferred mammalian host cells which exhibit recombinant antibodies of the invention comprise Chinese voles ovary (CHO cells) (comprising dhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77 : 4216-4220, It is used with DHFR selectable marker genes, as described in RJ Kaufman and PA Sharp (1982) Mol. Biol. 159 :601-621), NSO myeloma cells, COS cells and SP2 cells. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a time sufficient to express the antibody in the host cell, or better, the antibody is secreted into the medium in which the host cell is grown. Antibodies can be recovered from culture media using standard protein purification methods.

Host cells can also be used to produce intact antibody sites, such as Fab fragments or scFv molecule. It is to be understood that variations of the above described procedures are within the scope of the invention. For example, it may be desirable to transfect a host cell with DNA encoding one (but not both) of the light or heavy chain of an antibody of the invention. Recombinant DNA technology can also be used to remove some or all of the DNA encoding one or both of the light or heavy chains, which is not necessary for binding to hTNFα. Molecules exhibiting DNA molecules from this truncation are also included in the antibodies of the present invention. In addition, the antibody of the present invention can be cross-linked to a second antibody by standard chemical crosslinking methods to produce a bifunctional antibody. One heavy chain and one light chain are the antibodies of the present invention and other heavy and light chains are other than hTNFα. The antigen is specific.

In a preferred system for recombinant expression of an antibody or antigen binding site thereof, a recombinant expression vector encoding both an antibody heavy chain and an antibody light chain is introduced into dhfr-CHO cells by calcium phosphate regulatory transfection. In recombinant expression vectors, antibody heavy and light chain genes are each operably linked to a CMV enhancer/AdMLP promoter regulatory element to confer a high degree of gene transfection. The recombinant expression vector also carries the DHFR gene, which can select CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformed host cell culture allows for antibody heavy and light chain expression and recovery of intact antibodies from the culture medium. Standard biomolecular techniques are used to prepare recombinant expression vectors, transfect host cells, select transgenic strains, culture host cells, and recover antibodies from culture media.

III. Recombinant Human Antibody Selection

In addition to the D2E7 or antigen binding site thereof disclosed herein or the D2E7-related antibody, the recombinant human antibody of the present invention can be isolated by screening a recombinant antibody library, preferably using humans derived from mRNA derived from human lymphocytes. The scFv phage display library of VL and VH cDNA preparations. Methods of preparing and screening such databases are known in the art. In addition to producing the commercially available phage display library of sets other than the group (such as pharmaceutical Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP ^TM phage display kit, catalog number 240612), in particular for producing and correcting Examples of methods and reagents for screening antibody display databases can be found in, for example, Ladner et al., USP 5,223,409; Kang et al., PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/ 20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffery et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. 1991) Bio/Technology 9 : 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3 : 81-85; Hues et al. (1989) Science 246 : 1275-1281; McCafferty et al., Nature (1990) 348 :552 -554; Griffiths et al. (1993) EMBO J 12 : 725-734; Hawkins et al. (1992) J Mol Biol 226 : 889-896; Clackson et al. (1991) Nature 352 : 624-628; Gram et al. (1992). ) PNAS 89 : 3576-3580; Garrard et al. (1991) Bio/Technology 9 :1 373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19 : 4133-4137; and Barbas et al. (1991) PNAS 88 : 7978-7982.

In a preferred embodiment, in order to isolate a human antibody having high affinity and low dissociation constant for hTNFα, a mouse anti-hTNFα antibody having high affinity to hTNFα and a low dissociation constant (such as MAK 195, having the accession number ECACC) is first used. A fusion tumor of 87 050801) A human heavy and light chain sequence having similar hTNFα binding activity was selected by the method using the antigenic determining gene described in Hoogenboom et al. PCT Publication No. WO 93/06213. The antibody database used in this method is preferably McCafferty et al. PCT Publication No. WO 92/01047, McCafferty et al, Nature (1990) 348 :552-554; and Griffiths et al. (1993) EMBO J 12 :725-734. The scFv database prepared and screened as described. The scFv antibody library is preferably screened using recombinant human TNFα as an antigen.

Once the original human VL and VH fragments were selected, a "mix and pair" experiment was performed in which the initially selected VL and VH fragments of the hTNF[alpha] binding were screened differently to select the preferred VL/VH pairwise combination. In addition, in order to further improve the affinity and/or reduce the dissociation constant of hTNFα binding, the VL/VH paired VL and VH fragments may be similar to those in the body wall mutation process in which the antibody affinity mutation is responsible for the natural immune reaction. Random mutations, preferably in the CDR3 region of VH and/or VL. This in vitro affinity mutation can be performed by amplifying the VH and VL regions using PCR primers complementary to the VH CDR3 or VL CDR3, respectively, which have been spiked (spiked) in a random mixture of 4 nucleotide bases at certain positions. Thus, the resulting PCR product can encode VH and VL fragments in which a random mutation has been introduced into the VH and/or VL CDR3 region. The randomly mutated VH and VL fragments are then screened for binding to hTNF[alpha] and may select sequences that exhibit high affinity and low off rate for hTNF[alpha] binding.

After screening from the recombinant immunoglobulin display library and isolated from the anti-hTNFα antibody of the present invention, it can be returned from the display package (such as the autophagic genome). The nucleic acid encoding the selected antibody and the standard recombinant DNA technology can be subcloned into other expression vectors. If desired, the nucleic acid can be manipulated to produce additional antibody types of the invention (e.g., linked to nucleic acids encoding other immunoglobulin regions, such as other constant regions). To express a recombinant human antibody isolated by the screening pool, the DNA encoding the antibody is cloned into a recombinant expression vector and introduced into a mammalian host cell, as further detailed in paragraph II above.

IV. Pharmaceutical Compositions and Pharmaceutical Investment

The antibodies and antibody sites of the invention can be incorporated into pharmaceutical compositions suitable for administration to an individual by the methods described herein, such as biweekly subcutaneous administration. Typically, the pharmaceutical composition comprises an antibody (or antibody site) of the invention and/or methotrexate and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible and suitable for administration to an individual by the methods herein. Wait. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it is preferred to include an isotonic agent such as a saccharide, a polyol such as mannitol, sorbitol or sodium chloride in the composition. Pharmaceutically acceptable carriers can also include minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers which enhance the shelf life or efficiency of the antibody or antibody site.

The compositions of the invention can be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (such as injection and perfusion solutions), dispersions or suspensions, lozenges, pills, powders, liposomes, and suppositories. The preferred form depends on the mode of administration desired and the therapeutic use. A typical preferred composition is an injectable or injectable solution, such as is used in humans to passively immunize other antibodies. Composition. The preferred mode of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular injection. In a particularly preferred embodiment, the antibody is administered by subcutaneous injection (e.g., biweekly subcutaneous injection).

Therapeutic compositions generally require sterility and are stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome or other grade structure suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount, if necessary, in one or a combination of the above ingredients, in a suitable solvent, followed by filter sterilization. In general, the dispersion may be incorporated into the active ingredient in the active vehicle containing the active liquid carrier and the other ingredients required for the preparation. In the case of preparing sterile powders for sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization which yields the active ingredient powder plus any additional desired ingredients in any of the foregoing filter sterilizing solutions. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, such as monostearate and gelatin.

The antibodies and antibody sites of the invention can be administered by a variety of methods known in the art, but for many therapeutic uses, the preferred route/mode of administration is subcutaneous injection. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary with the desired result. In certain embodiments, the active compound can be prepared from carriers which will protect the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyethylene glycol (PEG), Polyanhydride, polyglycolic acid, collagen, polyorthoester and polylactic acid. Many methods of preparing such formulations are patented and generally known to those skilled in the art. See, for example, Continuous and Controlled Drug Delivery Systems, edited by J.R. Robinson, Marcel Dekker, New York, 1978.

In some embodiments, the benzene inventive antibody or antibody site can be administered orally, for example, with an inert diluent or a digestible carrier. The compound (and other ingredients if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into a lozenge or directly incorporated into the individual's diet. For oral therapeutic administration, the compounds can be incorporated into excipients and used in digestible troches, buccal tablets, troches, capsules, candies, suspensions, syrups, wafers and the like. In order to administer a compound of the invention parenterally, it is necessary to avoid coating the deactivated material or co-administering with the compound.

Supplementary active compounds can also be incorporated into the compositions. In some embodiments, an antibody or antibody site of the invention is co-administered and/or co-administered with one or more other therapeutic agents. For example, the anti-hTNFα antibody or antibody site of the present invention may be associated with methotrexate, one or more other antibodies that bind to other targets (eg, antibodies that bind to other cytokines or bind to cell surface molecules), one or more cytokines, and soluble TNFα. (for example, see PCT Bulletin No. WO 94/06476) and/or one or more chemical agents that inhibit the production or activity of hTNFα (as described in PCT Publication No. WO 93/19751, such as cyclohexane derivatives) Or co-administered. Furthermore, one or more antibodies of the invention can be used in combination with two or more of the foregoing therapeutic agents. This combination therapy may facilitate the administration of a lower dose of the therapeutic agent, thus avoiding possible toxicity or complications associated with various monotherapies. Combining the antibody or antibody site of the present invention and other therapeutic agents Further details are given in paragraph IV.

Non-limiting examples of therapeutic agents for rheumatoid arthritis that can be used in combination with an antibody or antibody site of the invention include the following: non-sterol anti-inflammatory agents (NSAIDs); cytokine inhibitory anti-inflammatory agents (CSAIDs); CDp-571/BAY-10 -3356 (humanized anti-TNFα antibody; Celltech/Bayer; cA2 (chemotaxis anti-TNFα antibody Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see eg Arthritis & Rheumatism ( 1994) Vol. 37 , S295; J. Invest. Med. (1996) Vol. 44 , 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (Non-clinded primate anti-CD4 antibody; IDEC/SmithKline; see, eg, Arthritis & Rheumatism (1995) Vol. 38 , S185); DAB 486-IL-2 and/or DBA 389-IL-2 (IL- 2 fusion protein; Seragen; see for example Arthritis & Rheumatism (1993) Vol. 36 , 1223); anti-Tac (humanized anti-IL-2Rα; Protein Design Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/) Schering); IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering); IL-4; IL-10 and/or IL-4 agonist (eg agonist antibody); IL -1RA (IL-1 receptor antagonist; Synergen/Amgen); TNF-bp/s-TNFR (soluble TNF-binding protein, see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplement) S284; Amer. J. Physiol.-Heart and Circulatory Physiology (1995) Vol. 268, pp. 37-42); R973401 (phosphodiesterase type IV inhibitor; see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplementary)) , S282); MK-966 (COX-2 inhibitor; see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplement), S81); Iloprost (see, for example, Arthritis & Rheumatism (1996) Vol. 39 , p. Phase 9 (additional version), S82); Aminoguanidine; Amidinopiperidone (see, for example, Arthritis & Rheumatism (1996), Vol. 39 , No. 9 (Supplement), S282) and imipenemone-related drugs (e.g., Celgen); leflunomide (anti-inflammatory and cytokine inhibitors, see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplement), S131; Inflammation Research (1996) Volume 45 , pp. 103-107); tranexamic acid (inhibitor of plasma activation; see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplement), S284); T- 614 (cytokine inhibitor; see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplementary), S282); prostaglandin E1 (see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplementary) ), S282); Tenidap (non-sterol anti-inflammatory drugs; see, for example, Arthritis & Rheumatism (1996), Vol. 39 , No. 9 (Supplement), S280); Naproxen (non-sterol) Anti-inflammatory drugs; see, for example, Neuro Report (1996) Vol. 7 , p. 1209-1213); Meloxicam (non-sterol anti-inflammatory drugs); Ibuprofen (non-sterol anti-inflammatory drugs); Piroxicam (non-sterol anti-inflammatory drugs); Diclofenac (non-sterol anti-inflammatory drugs); Indomethacin (non-sterol anti-inflammatory drugs); Sulfasalazine (see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplementary), S281); Azathioprine (see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplement), S281); ICE inhibitor (enzyme interleukin-1β converting enzyme); zap-70 and/or lck inhibitor (inhibitor of tyrosine kinase zap-70 or lck); VEGF inhibitor and/or VEGF-R inhibition Agent (vascular endothelial growth factor or vascular endothelial growth factor receptor; inhibitor of angiogenesis); corticosteroid anti-inflammatory drugs (such as SB203580); TNF-converting enzyme inhibitor; anti-IL-12 antibody; Algin-11 (see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplement), S296); Interleukin-13 (see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplementary) ), S308); interleukin-17 inhibitor (see, for example, Arthritis & Rheumatism (1996) Vol. 39 , No. 9 (Supplement), S120); gold; penicillamine; chloroquinoline; hydroxychloroquinoline; chlorobutyric acid Nitrogen mustard; cyclophosphamide; cyclosporine; total lymphoid radiation; anti-thymocyte globulin; anti-CD4 antibody; CD5-toxin; peptide and collagen for oral administration; lobenzarit disodium Cytokine Modulators (CRAs) HP228 and HP466 (Houghten Pharmaceuticals Inc.); ICAM-1 antisense phosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals); soluble complementary receptor 1 (TP10; T cell science company); prednisone; orgotein; glycosaminoglycan polysulfate; minocycline; -IL2R antibody; mice and plant lipids (fish and plant seed fatty acids; see DeLuca et al. (1995) Rheum.Dis.Clin.North e.g. Am 21:. 759-777); golden brown pigment (auranofin); phenylbutyrate hydrazone ; meclofenac; flufenamic acid; intravenous immunoglobulin; rilukone; mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amyprilose (therafectin); cladribine (2-chlorodeoxyadenosine); and azoribine.

Non-limiting examples of therapeutic agents for inflammatory bowel disease for use in combination with an antibody or antibody site of the invention include the following: budenoside; epidermal growth factor; corticosteroids; cyclosporine; sfafa well; amine-based water Salicylate; 6-hydrogenthioguanidine; azathiopurine; metronidazole; lipoxygenase inhibitor; mesalamine; olsalazine; balsalazide; Thromboxane inhibitor; IL-1 receptor antagonist; anti-IL-1β monoclonal antibody; anti-IL-6 monoclonal antibody; growth factor; elastase inhibitor; pyridyl imidazole compound; CDP-571/BAY -10-3356 (humanized anti-TNFα antibody; Celltech/Bayer; cA2 (chemotaxis anti-TNFα antibody Centocor); 75 kd TNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see for example Arthritis & Rheumatism ( 1994) Vol. 37 , S295; J. Invest. Med. (1996) Vol. 44 , 235A); 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); interleukin-10 (SCH 52000) ;Schering Plough); IL-4; IL-10 and / or IL-4 agonist (such as agonist antibody); interleukin-11; Panison , dexamethasone or budaside glucuronide- or dextran-conjugated prodrug; ICAM-1 antisense phosphorothioate oligodeoxynucleotide (ISIS 2302; Isis Pharmaceuticals Inc. Soluble Complementary Receptor 1 (TP10; T Cell Science); low-release mexican well; methotrexate; antagonist of platelet activating factor (PAF); ciprofloxacin; and lidocaine.

Non-limiting examples of therapeutic agents for multiple sclerosis that can be combined with an antibody or antibody site of the invention include the following: corticosteroids; pannisone; methyl dehydropannisone; azathiane; cyclophosphamide; cyclosporine Su; methotrexate; 4- aminopyridine; for xanthone piperidine (tizanidine); interferons -β1a (Avonex ^TM; Biogen); interferons ^{-β1b (Betaseron TM; Chiron / Berlex} ); copolymer 1 (Cop-1; Copaxone ^TM ; Teva Pharmaceuticals; high specific gravity oxygen; intravenous immunoglobulin; crabibine; CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer); cA2 (chimeric anti- TNFα antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see, eg, Arthritis & Rheumatism (1994) Vol. 37 , S295; J. Invest. Med. (1996) Vol. 44 , 235A) 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche); IL-10; IL-4; and IL-10 and/or IL-4 agonist (eg agonist antibody).

Non-limiting examples of therapeutic agents for sepsis that can be combined with an antibody or antibody site of the invention include the following: an isotonic saline solution; an antibiotic; intravenous gamma globulin; continuous blood filtration; carbapenems (eg, merlotine) Meropenem)); cytokine antagonists such as TNFα, IL-1β, IL-6 and/or IL-8; CDP-571/BAY-10-3356 (humanized anti-TNFα antibody; Celltech/Bayer); cA2 ( Chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see, eg, Arthritis & Rheumatism (1994) Vol. 37 , S295; J. Invest. Med. (1996) 44, 235A); 55 kdTNFR- IgG (55 kD TNF receptor -IgG fusion protein; Hoffmann-LaRoche); cytokine regulating agents (CRAs) HP228 and HP466 (Houghten pharmaceutical company); SK & F 107647 (low molecular peptide; SmithKline Beecham ; tetravalent guanidine group CNI-1493 (Picower Association); tissue factor pathway inhibitor (TFPI; Chiron); PHP (chemically modified erythropoietin; APEX Bioscience); iron chelator and chelate, including diexi Triamine pentaacetic acid-iron (III) complex (DTPA iron (III); Molichem medicine); lysine film (liso Fylline) (synthesis of small molecule methylxanthine; cell therapy company); PGG-dextran (aqueous soluble beta 1,3-glucan; Alpha-Beta Technologies); lipid-reconstituted apolipoprotein A- 1; palmitic hydroxydecanoic acid (synthetic antibacterial agent can inhibit lipid A biosynthesis); anti-endotoxin antibody; E5531 (synthetic lipid A antagonist; Eisai, USA); rBPI ₂₁ (human bactericidal / increase permeability protein) Recombinant N-terminal fragment); and synthetic anti-endotoxin peptide (SAEP; BioYnth Research Laboratories).

Non-limiting examples of therapeutic agents for adult respiratory distress syndromes that can be combined with an antibody or antibody site of the invention include the following: anti-IL-8 antibody; surfactant replacement therapy; CDP-571/BAY-10-3356 (humanized antibody -TNFα antibody; Celltech/Bayer); cA2 (chimeric anti-TNFα antibody; Centocor); 75 kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein; Immunex; see, eg, Arthritis & Rheumatism (1994), Vol. 37 , S295 J. Invest. Med. (1996) Vol. 44 , 235A); and 55 kdTNFR-IgG (55 kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche).

The compositions of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antibody site of the invention. "Therapeutically effective amount" means an amount effective to achieve the desired therapeutic result during administration and for the required period of time. Antibody or antibody The therapeutically effective amount can be determined by a number of factors, such as the individual's disease state, age, sex, and weight, and the ability of the antibody or antibody portion to elicit the desired response in the individual. A therapeutically effective amount can also be one in which the therapeutic benefit of the antibody or antibody site is greater than any toxic or deleterious effect. "Prophylactically effective amount" means the amount of preventative result that can be effectively achieved during the administration and the required period of time. Typically, since the prophylactic dose is administered to the individual at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The medication regimen can be adjusted to provide the optimal response (eg, treatment or prophylaxis). For example, a single pellet can be administered, and several divided doses can be administered over a period of time, or the dose can be proportionally reduced or increased, as indicated by the urgency of the therapeutic standpoint. Formulating a parenteral composition in unit dosage form is particularly advantageous in terms of ease of administration and uniformity of dosage. Dosage unit form as used herein refers to a unit that is physically separated by unit dosages for the individual to be treated; each unit contains the active compound which is in a The unit dosage form of the invention is subject to and directly related to: (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the field of mixing the active compound for the treatment of the individual's inductivity. .

A therapeutically or prophylactically effective amount of an antibody or antibody site of the invention is, without limitation, from 10 to 100 mg, more preferably from 20 to 80 mg, and most preferably 40 mg. It should be noted that the dose quality may vary depending on the type and severity of the condition to be soothed. It is also to be understood that for any particular individual, the particular course of treatment will be adjusted over time according to the individual needs and the professional judgment of the administering or monitoring composition, and the dosage ranges described herein are merely exemplary and not intended to limit the present. Scope of the invention or operating.

V. Use of the antibody of the present invention

Given the ability to bind to hTNFα, the anti-hTNFα antibody of the present invention or a portion thereof can be subjected to conventional immunoassay such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry. The method detects hTNFα (as in a biological sample such as serum or plasma). The present invention provides a method for detecting hTNFα in a biological sample, comprising contacting a biological sample with an antibody of the present invention or an antibody site thereof, and detecting an antibody (or antibody site) or an unbound antibody (or antibody site) that binds to hTNFα. . The antibody is labeled, directly or indirectly, with a detectable substance to accelerate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetaminocholine; examples of suitable prosthetic complexes include streptavidin/biotin and avidin/biotin Examples of suitable fluorescent materials include umbelliferone, luciferin, fluorescein isothiocyanate, alkaline ruthenium, dichlorotriamine fluorescein, 5-dimethylamine-1- Naphthalene sulfonium chloride or phycoerythrin; examples of luminescent materials comprising luminol; and examples of suitable radioactive materials comprising ¹²⁵ I, ¹³¹ I, ³⁵ S or ³ H.

In addition, the antibody is labeled, and hTNFα can be analyzed in a biological fluid by a competitive immunoassay using a detectable substance-labeled rhTNFα substance and an unlabeled anti-hTNFα antibody. In this assay, the biological sample, the labeled rhTNFα standard, and the anti-hTNFα antibody can be mixed and assayed for the amount of labeled rhTNFα standard bound to the unlabeled antibody. The amount of hTNFα in the biological sample is inversely proportional to the amount of labeled hTNFα standard bound to the anti-hTNFα antibody.

The D2E7 antibody of the present invention can also be used for detecting TNFαs derived from septicemia other than humans, especially from primates (such as chimpanzees, baboons, baboons, baboons and rhesus monkeys), pigs and mice, because D2E7 It can bind to each of these TNFαs.

The antibodies and antibody sites of the invention can neutralize hTNFα activity in vitro and in vivo (see USP 6,090,382). Furthermore, at least some of the antibodies of the invention, such as D2E7, neutralize hTNFα activity from other species. Accordingly, the antibody and antibody sites of the present invention can be used, for example, in cell cultures containing hTNFα, human subjects, or other mammalian individuals having TNFαs that can cross-react with the antibodies of the present invention (e.g., chimpanzees, baboons, baboons, baboons, icy Hippocampus, pigs and mice) inhibited hTNFα activity. In one embodiment, the invention provides a method of inhibiting TNFα activity comprising contacting TNFα with an antibody or antibody site of the invention thereby inhibiting TNFα activity. Preferably, the TNFα is human TNFα. For example, in a cell culture containing or suspending TNFα, the antibody or antibody site of the present invention can be added to the culture medium to inhibit hTNFα activity in the culture.

In a preferred embodiment, the invention provides a method of treating a disorder in which administration of an anti-TNFa antibody is beneficial, comprising subcutaneously administering an antibody or antibody site of the invention to a subject for treatment, thereby treating the disorder. In a particularly preferred embodiment, the anti-system is administered subcutaneously in a biweekly manner. In another particularly preferred embodiment, the anti-system is administered subcutaneously before, during or after methotrexate administration. Preferably, the individual is a human individual. Alternatively, the individual can be a mammal having TNF[alpha] that can cross-react with an antibody of the invention. Still other individuals may be mammals in which hTNF[alpha] has been introduced (e.g., by administration of hTNF[alpha] or by hTNF[alpha] gene transfer). this invention Antibodies can be administered to human subjects for therapeutic purposes (as discussed above). Furthermore, the antibodies of the invention may be administered to non-human mammals (e.g., primates, pigs or mice) having TNF[alpha] that are cross-reactive with the antibodies of the invention for veterinary purposes or as an animal model of human disease. Regarding the latter, this animal model can be used to evaluate the therapeutic effects of the antibodies of the invention (e.g., test dose and time course of administration).

As used herein, "a disorder in which administration of an anti-TNFα antibody is beneficial" is intended to encompass diseases and other disorders in which an individual having the disorder has been shown to be or is expected to be responsible for the pathology of the disorder or a disorder causing the disorder. Or other anti-TNFα antibodies or biologically active sites thereof have been shown to be successful in treating the disease. Accordingly, a disorder in which TNFα activity is detrimental is a condition in which inhibition of TNFα is expected to soothe the symptoms and/or progression of the disorder. This disorder can be demonstrated by, for example, an increase in the concentration of TNFα in the biological fluid of the individual suffering from the disorder (e.g., serum, plasma, synovial fluid, etc. of the individual), which can be detected using, for example, the anti-TNFα antibody described above. There are several examples of disorders in which TNFα activity is harmful. The use of the antibodies and antibody sites of the invention in the treatment of specific diseases is further discussed below:

A. Septicemia

Tumor necrosis factor has established a role in the pathology of sepsis, and biological effects include hypotension, myocardial depression, vascular leakage syndrome, organ necrosis, stimulation of the release of toxic minor regulators, and activation of the clot cascade Role (see, for example, Tracey, KJ and Cerami, A. (1994) Annu. Rev. Med. 45 : 491-503; Russell, D and Thompson, RC (1993) Curr. Opin. Biotech. 4 : 714-721). Accordingly, the human antibodies and antibody sites of the invention can be used to treat any clinically defined sepsis, including septic shock, endotoxic shock, Gram-negative sepsis, and toxic shock syndrome.

Furthermore, for the treatment of sepsis, the anti-hTNFα antibody or antibody site of the invention may be administered in combination with one or more other therapeutic agents that can relieve sepsis, such as interleukin-1 inhibitors (eg PCT Publication No. WO 92/16221) And WO 92/17583), cytokine interleukin-6 (see, for example, PCT Publication No. WO 93/11793) or platelet activating factor antagonist (see, for example, European Patent Application Publication No. EP 374 510).

Further, in a preferred embodiment, the anti-TNFα or antibody site of the present invention is administered to a human subject, and the subgroup of the end septicemia group has a serum or plasma concentration of IL-6 higher than 500 pg/ml at the time of treatment. And more preferably 1000 pg/ml (see Daum, L et al. PCT Bulletin No. WO 95/20978).

B. Autoimmune disease

Tumor necrosis factor is associated with roles in the pathology of various autoimmune diseases. For example, TNFα is associated with inflammation of activated tissues and joint destruction that causes rheumatoid arthritis (see, for example, Tracey and Cerami, supra; Arend, WP and Dayer, JM. (1995) Arth. Rheum. 38 :151-160; Fava, RA, et al. (1993) Clin. Exp. Immunol. 94 :261-266). TNF[alpha] is associated with promoting island cell death and modulating diabetic insulin resistance (see, for example, Tracey and Cerami, supra; PCT Publication No. WO 94/08609). TNFα is also associated with modulation of cytotoxicity in oligodendrocytes and induction of inflammatory plaques in multiple sclerosis (see, for example, Tracey and Cerami, supra). Embedding and humanized mouse anti-hTNFα antibodies have undergone clinical testing for the treatment of rheumatoid arthritis (see, for example, Elliott, MJ, et al. (1994) Lancet 344 : 1125-1127; Elliot, MJ, et al. (1994) Lancet 344 : 1105-1110; Rankin, EC, et al. (1995) Br. J. Rheumatol. 34 : 334-342).

The human antibody and antibody site of the present invention can be used for treating autoimmune diseases, especially those associated with inflammation, including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gout arthritis, allergy, multiple sclerosis, autoimmune diabetes, Automated immunization with uveitis and kidney disease syndrome. Typically, the antibody or antibody site is administered systemically, but for certain disorders, topical administration of antibodies or antibody sites at the site of inflammation may be beneficial (eg, topical administration of rheumatoid arthritis or ulceration of diabetes) Topical administration can be administered alone or in combination with a cyclocyclohexylene derivative as described in PCT Publication No. WO 93/19751).

C. Infected diseases

Tumor necrosis factor is associated with the physiological effects seen in the regulation of various infectious diseases. For example, TNFα is associated with regulation of brain inflammation and capillary embolism and infarction in malaria (see, for example, Tracey and Cerami, supra). TNFα is also associated with regulation of brain inflammation, including blood-brain barrier rupture of meningitis, restriction of septic shock syndrome, and activation of venous infarction (see, for example, Tracey and Cerami, supra). TNFα is also associated with the inclusion of malignant diseases, acquired immune deficiency syndrome (AIDS) in stimulating viral proliferation, and modulation of central nervous system damage (see, for example, Tracey and Cerami, supra). Accordingly, the antibodies and antibody sites of the invention can be used to treat infectious diseases, including bacterial meningitis (see, for example, European Patent Publication No. EP 585 705), cerebral malaria, AIDS, and AIDS-related complications (ARC) (see, for example, European Patent Ref. EP 230 574) and secondary cytomegalovirus infections for transplantation (see, for example, Fietze, E., et al. (1994) Transplant 58: 675-680). The antibody and antibody sites of the present invention can also be used to alleviate the symptoms associated with infectious diseases, including fever and myalgia caused by infections (such as influenza) and septic diseases such as recurrent AIDS or ARC.

D. Transplant

Tumor necrosis factor is associated with graft rejection and a major regulator of graft-to-host disease (GVHD) and is associated with inhibition of renal transplant rejection when the rat antibody OKT3 associated with the anti-T cell receptor CD3 complex is used to inhibit renal transplant rejection. For the adjustment of the reaction (see, e.g. Tracey and Cerami, literature supra; Eason, JD, et al. (1995) transplantation 59: 300-305; Suthanthiran, M and Strom, TB, (1994) New Engl.J.Med 331.. :365-375). Accordingly, the antibodies and antibody sites of the invention can be used to inhibit transplant rejection, including allografts and allografts, and inhibition of GVHD. Although the antibody or antibody site can be used alone, it is preferably used in combination with one or more other drugs that inhibit the immune response against allogeneic transplantation or inhibit GVHD. For example, in one embodiment, an antibody or antibody site of the invention can be used in combination with OKT3 to inhibit OKT3-induced responses. In another embodiment, an antibody or antibody site of the invention is used in combination with one or more antibodies directed to other targets that modulate an immune response, such as the cell surface molecule CD25 (interleukin-2 receptor-alpha), CD11a (LFA- 1), CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86 (B7-2). In yet another embodiment, the antibody or antibody site of the invention is used in combination with one or more general immunosuppressive agents such as cyclosporin A or FK506.

E. Malignant disease

Tumor necrosis factor is associated with malignant disease-induced malignant disease, stimulation of tumor growth, enhancement of migration potential, and regulation of cytotoxicity (see, for example, Tracey and Cerami, supra). Accordingly, the antibodies and antibody sites of the invention can be used to treat malignant diseases to inhibit tumor growth or migration and/or to alleviate the malignant disease of malignant disease. The antibody or antibody site can be administered systemically or locally to the tumor site.

F. Lung disorders

Tumor necrosis factor is associated with the pathology of adult respiratory distress syndrome, including stimulation of leukocyte-endothelial cell activation, introduction of cytotoxicity into lung cells, and induction of vascular leakage syndrome (see, for example, Tracey and Cerami, supra). Accordingly, the antibodies and antibody sites of the invention can be used to treat various lung disorders, including adult respiratory distress syndrome (see, for example, PCT Bulletin No. WO 91/04054), pulmonary shock, chronic pulmonary inflammatory disease, pulmonary sarcoma, pulmonary fibrosis, and Bauxite deposits. The antibody or antibody site can be administered systemically or locally to the surface of the lung, for example, as an aerosol.

G. intestinal disorders

Tumor necrosis factor is associated with the pathology of inflammatory bowel disease (see, for example, Tracey, KJ, et al. (1986) Science 234 : 470-474; Sun, XM., et al. (1988) J. Clin. Invest. 81 :1328 -1331; MacDonald, TT, et al. (1990) Clin. Exp. Immunol. 81 :301-305). Embedding mouse anti-hTNFα antibodies has been clinically tested for Cologne's disease (van Dullemen, HM, et al. (1995) Gastroenterology 109 : 129-135). The human antibodies and antibody sites of the invention can also be used to treat intestinal disorders such as idiopathic inflammatory bowel disease, which include two syndromes: Cologne's disease and colon ulcer.

H. Heart disorder

The antibodies and antibody sites of the invention can also be used to treat a variety of cardiac disorders, including cardiac anemia (see, for example, European Patent Publication EP 453 898) and cardiac insufficiency (cardiac muscle weakness) (see, for example, PCT Publication No. WO 94/20139).

I. Other

The antibodies and antibody sites of the invention can also be used to treat a variety of other disorders in which TNF[alpha] activity is detrimental. Examples of other diseases and disorders in which TNFα activity is associated with pathology and which can be treated using the antibodies or antibody sites of the invention include inflammatory bone disorders and bone resorption diseases (see, for example, Bertolini, DR, et al. (1986) Nature 319 :516-518 ;Konig, A., et al. (1988) J. Bone Miner. Res. 3 :621-627; Lerner, UH and Ohlin, A. (1993) J. Bone Miner. Res. 8 : 147-155; and Shankar , G. and Stern, PH (1993) Bone 14 : 871-876), hepatitis contains alcoholic hepatitis (see, for example, McClain, CJ and Cohen, DA (1989) Hepatology 9 : 349-351; Felver, ME, et al. (1990) Alcohol. Clin. Exp. Res. 14 : 255-259; and Hansen, J., et al. (1994) Hepatology 20 : 461-474) and viral hepatitis (Sheron, N., et al. (1991). Journal of Hepatology 12:241-245; and Hussain, MJ, et al. (1994) J. Clin. Pathol. 47 : 1112-1115), coagulation interference (see, for example, van der Poll, T., et al. (1990) N. Engl. J. Med. 322 : 1622-1627; and van der Poll. T. et al. (1991) Prog. Clin. Biol. Res. 367 : 55-60), burns (see, for example, Giroir, BP, etc.) . al (1994) Am.J Physiol 267:. H118-124; and Liu, XS, et al. (1994) burns 20: 40-44), and then Note damage (see, e.g. Scales, WE, et al. (1994) Am.J.Physiol 267:.. G1122-1127; Serrick, C, et al. (1994) Transplantation 58: 1158-1162; and Yao, YM, et al. (1995) Resucitation 29 : 157-168), neoplasia (see, for example, McCauley, RL (1992) J. Clin. Immunol. 12 : 300-308), scar tissue formation, and fever.

The invention is further illustrated by the following examples, which should not be construed as limiting. All references, patents and publications cited in this specification are hereby incorporated by reference.

Example 1 : Treatment with anti-TNFα antibody

D2E7 efficiency after subcutaneous administration

In this study, 24 patients with RA were treated with s.c. injections for 3 months per week at a dose of 0.5 mg/kg D2E7 (n=18) or placebo (n=6). The average age of the patients who participated in the study was 10.1 years and the disease index (DAS) was 4.87 and the average 3.4 DMARDs (disease-modifying anti-rheumatic drugs); again reflected the equivalent disease activity. The sensor continued to be treated with D2E7 open-labeling, while patients who did not respond to the 0.5 mg/kg dose or who lost the DAS response to the 0.5 mg/kg dose were given a s.c. injection of 1 mg/kg at the 12th week of the study.

The first group of patients was enrolled for up to 60 injections and the study drug was therefore accepted for 60 weeks. Up to 78% of patients achieved DAS and ACR20 responses during the first week of treatment. Subcutaneous administration of D2E7 at a dose of 0.5 mg/kg/week reduced the number of joint swelling (SWJ), the number of softened joints (TJC) by 61%, and CRP at 12 weeks. 39% (compared to the baseline), while all parameters in the placebo group increased. After completion of the placebo control during the study period, the patient continued to be treated with continued efficiency for 14 months. These results therefore show that subcutaneous administration of D2E7 at a dose of 0.5 mg/kg/week can safely self-administer the drug with good local tolerance.

D2E7 and methotrexate administration

In this study, placebo or D2E7 was administered at a dose of 1 mg/kg sc or iv, except for the start of treatment with (metamidine) MTX. 54 patients were included in the study and 18 patients received iv for D2E7 and sc Placebo, 18 patients received placebo and sc received D2E7 and 18 iv and sc received placebo. The patient received the second dose only after losing his blind response, no earlier than 4 weeks after the first dose. Subsequently, all patients received an open label for biweekly s.c. injection of D2E7.

The demographic characteristics of the study included an average RA period of 11.1 years, an average of 3.6 DMARDs before exposure (non-MTX), and an average DAS of 4.81 when included in the study. On day 29, 72% of i.v.D2E7 treated patients and 44% of s.c.D2E7 treated patients had reached the DAS standard response compared to only 28% of placebo-treated patients (described in Figure 5). Of the participants in the study, 28% of placebo-treated patients maintained ACR20 response on day 29 compared to 72% of iv-treated D2E7 patients and 67% of sc-treated D2E7 patients at 1 and 3 months. Maintain its response within.

Example 2 : Total human dose of anti-TNFα antibody administered subcutaneously

Subcutaneous administration of D2E7 every week

This study included 284 patients with RA and was designed to determine the optimal total human dose for subcutaneous administration of D2E7. Patients receive 20, 40 or 80 mg per week at random D2E7 or placebo for 12 weeks, followed by placebo treatment blindly switched to 40 mg D2E7/week.

Approximately 49% of patients had ACR20 at 20 mg, 55% of patients had ACR20 at 40 mg and 54% of patients had ACR20 at 80 mg, and only 10% received placebo patients with ACR20 (described in Figure 1A). Approximately 23% of patients had ACR50 at 20 mg, 27% had ACR50 at 40 mg, and 20% had ACR50 at 80 mg, and only 2% received placebo for ACR50. These data indicate that subcutaneous administration of D2E7 (especially at a dose of 40 mg/week) produces a good response.

Example 3 : Biweekly subcutaneous administration of anti-TNFα antibody

Biweekly subcutaneous administration of D2E7

Clinical efficacy, safety, immunogen, and tolerance to RA patients with potential MTX response were investigated by subcutaneous (s.c.) subcutaneous (S.c.) injections of placebo or D2E7 for several weeks at several doses.

Research design

A placebo-controlled, double-blind, randomized, rehospital study was performed on RA patients with insufficient MTX efficiency or tolerance. During the trial, the patient's sustained MTX dose and dose range were included in the specific inclusion criteria below.

The study consisted of two parts: 1) “Washing period” 4 weeks prior to the first dose of the drug, during which DMARDs were excluded (except for MTX); and 2) “Placebo-controlled period” during which patients were randomly assigned to 67 One quarter of patients received 20, 40 or 80 mg of D2E7 (total human dose) administered every other week for 24 weeks. Each study drug dose was administered by two s.c. injections of 1.6 ml each. The first dose of the patient is administered by a medical practitioner as part of the patient training. With The post-dose was self-administered by the participating patients under the direct surveillance of the first 4 weeks. Subsequently, the dose is administered by the patient, the patient designated by the patient, or by a medical professional outside the study site. Dosing for 4 or 5 weeks was assigned after each clinical evaluation. Patients were continuously examined at 1, 2, 3, 4, 6, 8, 12, 16, 20, and 24 weeks of the study, and joint examinations were performed by unknown evaluators, independent of the treating physician.

This study included 271 RA patients. The study population is representative of the moderate to severe RA population in North America: approximately 70% of women and most of them over 40. The population is selected using predetermined inclusion and exclusion criteria (as known to those skilled in the art), such as patients requiring RA diagnosis, such as the 1987-Revised American College Rheumatology (ACR) standard (described in Appendix A).

result

Figure IB and Figures 2-4 show that subcutaneous administration of D2E7 with ampicillin for two weeks is significantly better than placebo in reducing RA signals and signs after 24 weeks. All three D2E7 dose effects were statistically significantly better than placebo administered weekly. Furthermore, 40 mg and 80 mg D2E7 have superior effects than the 20 mg dose.

Equivalent

It will be apparent to those skilled in the art that <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; This equivalent is intended to be included in the scope of the present invention.

RA's ACR definition

Classification tree criteria and functions for rheumatoid arthritis (RA) in 1987

. If the patient is included in the 5RA subgroup listed in Table 7, and especially the physician is clinically diagnosed with RA, the patient is said to have RA. Standards 1, 2 and 3 need to last for at least 6 weeks.

Arthritis & Rheumatism, Vol. 31, No. 3 (March 1988)

Figure 1A and Figure IB show that patients with rheumatoid arthritis (RA) were administered subcutaneously with antibody D2E7 for 12 weeks (1A) or subcutaneously with antibody D2E7 and methotrexate for 24 weeks (1B). College Rheumatology 20 (ACR20) and ACR50 reactions. These data show that administration every other week is as effective as weekly dosing.

Figure 2 shows that patients with RA are administered subcutaneously with antibody D2E7 and ammonia every other week. ACR20, ACR50 and ACR70 were reacted 24 weeks after onychomycosis.

3A and 3B show the elapsed time course of a weak joint count (3A) and an expanded joint count (3B) at 24 weeks after subcutaneous administration of D2E7 and methotrexate subcutaneously in a patient with RA within 24 weeks.

Figure 4 shows the results of a short-term health review (SF-36) 24 weeks after subcutaneous administration of D2E7 and methotrexate in patients with RA. RP, physiological role; PF, physiological function; BP, physical pain; GH, general health; V, vitality; SF, social function; RE, emotional role; and ME, emotional health.

Figure 5 shows the percentage of ACR responders after single intravenous injection of antibody D2E7 and methotrexate in patients with RA.

Appendix A

Sequence number 1:

Sequence number 2:

Sequence number 3:

Appendix B

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<110> Bermuda's Abbott Biotech

<120> Method for administering an anti-TNF- α antibody

<150> US 60/296961

<151> 2001-06-08

<160> 37

<170> PatentIn Ver.2.0

<210> 1

<211> 107

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 121

<212> PRT

<213> Homo sapiens

<400> 2

<210> 3

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Variable Light Chain CDR3

<220>

<221> Variation

<222> (9)

<223> Xaa=Thr or Ala

<400> 3

<210> 4

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> Variable Heavy Chain CDR3

<220>

<221> Variation

<222> (12)

<223> Xaa=Tyr or Asn

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<210> 5

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<400> 5

<210> 6

<211> 17

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<210> 11

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<400> 30

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<211> 321

<212> DNA

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<400> 37

Claims (4)

Use of a pharmaceutical composition for the preparation of a medicament for the treatment of Cologne's disease, wherein the medicament is administered subcutaneously every 13 to 15 days to treat the disease, and wherein the pharmaceutical composition comprises a 40 mg dose of human anti- TNF α antibody, wherein the anti-TNF [alpha] antibody comprises a human IgG1 heavy chain constant region; variable light chain (V _L) region, which comprises a SEQ ID NO: CDR1 amino acid sequences shown in Figure 7, having SEQ ID NO: CDR2 of the amino acid sequence shown by 5 and CDR3 having the amino acid sequence of SEQ ID NO: 3; and a variable heavy chain ( _VH ) region comprising the gene shown by SEQ ID NO: The CDR1 of the amino acid sequence has the CDR2 of the amino acid sequence of SEQ ID NO: 6 and the CDR3 of the amino acid sequence of SEQ ID NO: 4. Use of a pharmaceutical composition for the preparation of a medicament for treating a colon ulcer, wherein the medicament is administered subcutaneously every 13 to 15 days to treat the disease, and wherein the pharmaceutical composition comprises a 40 mg dose of human anti-TNF [alpha] antibody, wherein the anti-TNF [alpha] antibody comprises a human IgG1 heavy chain constant region; variable light chain (V _L) region, which comprises a SEQ ID NO: CDR1 amino acid sequences shown in Figure 7, having the SEQ ID NO: CDR2 of the amino acid sequence shown by 5 and CDR3 having the amino acid sequence of SEQ ID NO: 3; and variable heavy chain ( _VH ) region comprising the amine having SEQ ID NO: The CDR1 of the acid sequence, the CDR2 having the amino acid sequence of SEQ ID NO: 6, and the CDR3 having the amino acid sequence of SEQ ID NO: 4. The use according to claim 1 or 2, wherein the _VL region of the human anti-TNFα antibody has the amino acid sequence of SEQ ID NO: 1 and the V _H region of the human anti-TNFα antibody has the SEQ ID NO: The amino acid sequence of 2. The use of claim 1 or 2, wherein the drug is administered to an individual having an immune response to a chimeric or humanized anti-TNFα antibody.